The History of Cystic Fibrosis by Dr James Littlewood OBE

Edited and produced by Daniel Peckham

 

1944 Howard PJ. Familial character of fibrocystic disease of the pancreas. Am J Dis Child 1944; 68:330-332.
Philip Howard of Detroit reviewed the previous twelve instances of fibrocystic disease of the pancreas where a familial occurrence had been reported including instances where both or only one of twins had been affected. He reported an unusual number of infant deaths in two families in which proven CF existed and suggested the family furnished an instance of heterozygous inheritance (see Andersen and Hodges, 1946 below).


This is the first suggestion that CF was an inherited disorder - later this was established by Andersen and Hodges (1946 below)

 

1944 Fanconi G, Botsztejn A. Familial pancreatic fibrosis with bronchiectasis. Schweiz Med Wchnschr 1944; 74:85. (German).
Fanconi’s 1936 report is considered by some Europeans to be the first clear description of cystic fibrosis (Fanconi et al, 1936 above). Here the authors described the family trees of 25 children with fibrocystic disease of the pancreas. They considered, incorrectly at it turned out, that recessive transmission was unlikely as there was consanguinity in only 3 of their cases and the proportion of affected siblings appeared too high. So they postulated the cause to be an early intrauterine toxic factor akin to haemolytic disease of the newborn.


Andersen & Hodges, 1946 (below) were the first to correctly identify the Mendelian recessive mode of transmission, although a heterozygous mode had been suggested as likely by Howard in 1944 (above).

 

1945 Wissler H, Zollinger HU. Familial congenital cystic fibrosis of pancreas with bronchiectasis. Helv Paediatr Acta 1945; 1 (Suppl 1):3-88.
This paper from Zurich describes the clinical and pathological features of CF noting that lung changes may be absent in “nurslings with pancreatic fibrosis who die early”. The cystic pancreatic changes often are only detected by microscopy and may be overlooked. “It seems highly probable that the inflammatory changes in the pancreas, biliary passages and bronchial walls are the result of a single process… probably due to intrauterine damage of maternal origin… no reasons for accepting a primary avitaminosis as the cause and a recessive hereditary disease is improbable”.

 

These Swiss authors mistakenly considered an hereditary cause of CF to be unlikely and suggested the cause was more likely due to intrauterine damage as had Fanconi & Botsztejn, 1944 (above).

 

1946 Andersen DH, Hodges RC. Celiac syndrome V. Genetics of cystic fibrosis of the pancreas with consideration of the etiology. Am J Dis Child 1946; 72:62-80.
Investigating 47 of their own families and 56 from the literature, the authors concluded that the familial incidence indicated a hereditary disorder with a recessive mode of inheritance, but which required more than one factor for its expression. Although the incidence in siblings approximates to the 25% expected of a Mendelian recessive trait, the authors observed that an hereditary condition requires more than one factor for its expression.

 

This was a quite different explanation to that given by Fanconi et al (1944) but in line with that of Bodian, 1952 (below). Andersen continued to believe that “the pulmonary infection is the result of the nutritional deficiency” – obviously she considered this to be the additional factor required.
However, this is the first clear statement, backed by clinical evidence, that CF is inherited in a Mendelian recessive manner. Others, including David Lawson in the UK, would also speculate on the relative contribution of the basic defect and the secondary effects of the defect (such as chest infections and malnutrition) to the ultimate outlook for the patient. i.e. if the secondary effects could be prevented would the outlook be much better? Subsequent progress showed that this was indeed the case in view of the steady improvement in outlook that occurred before modification of the basic defect was achieved.

 

1949 Lowe CU, May CD, Reed SC. Fibrosis of pancreas in infants and children: statistical study of clinical and hereditary features. Am J Dis Child 1949; 78:349-374. [PubMed]
These authors from the University of Minnesota reviewed the hereditary aspects of 134 patients with CF seen over 10 years at the Infant's and Children’s Hospital, Boston where previously May had worked. There were 118 sibships and the authors concluded that the condition was determined by a recessive gene as had Andersen and Hodges (Andersen & Hodges, 1946 above). They noted good weight gain before first symptoms and fair gains thereafter in those who survived. In discussing the clinical features they noted 44 children showed osteoporosis and retarded bone age. They questioned the importance of vitamin A deficiency in the pathogenesis as had others.

This is one of the earliest mentions of osteoporosis in people with CF – a complication that was to become a major problem for adults with CF in the years to come.

 

1952 Bodian ML. Fibrocystic Disease of the Pancreas. A Congenital Disorder of Mucus Production (Mucosis). London, W Heinemann, 1952.
The first substantial work on CF from the UK by Martin Bodian and his colleagues at the Hospital for Sick Children, Great Ormond Street, London (GOS). Dr. Martin Bodian (1910-1963), had been appointed morbid anatomist at GOS in 1946. Dr Archie Norman was the paediatrician in charge of the CF clinic at GOS and a leading figure in CF care in the UK for many years. Dr. Cedric Carter (1917-1984) was a clinical geneticist who started the first genetic counselling clinic at GOS in 1957.


In the preface of the book Martin Bodian notes the contents encompass almost a decade of work since Donald Paterson diagnosed the first child with CF at Great Ormond Street in 1943. The authors reviewed their own and previously published families and concluded there was a recessive mode of inheritance and suggested parents of a child with CF should be given a 1 in 4 risk of further children being affected; this confirmed the findings of Andersen & Hodges (1946 above) and the earlier suggestion of Howard (1944 above).

 

1962 Pileggi A. Cystic Fibrosis in a negro. Del Med J 1962; March:97-98.[PubMed]
A black infant admitted with bronchopneumonia at 8 months was diagnosed by three positive sweat tests as having cystic fibrosis. He died after 37 days. At autopsy, in addition to bronchopneumonia, the pancreas showed typical changes of cystic fibrosis. The authors note that at the Babies Hospital New York only 2 children of 397 cases were Negroes – an incidence of approximately 0.5% (di Sant’Agnese Am J Med 1956; 21:406).

 

1963 Batten J, Mir D, Simon G, Carter C. The prevalence of respiratory disease in heterozygotes for the gene for fibrocystic disease of the pancreas. Lancet 1963; i: 1348-1350. [PubMed]
The incidence of chronic bronchitis in parents of people with CF (obligate heterozygotes) and controls was similar to that reported for the general population – which was remarkably high at that time being 17% of men aged 40-60 years and 8% in women. The authors concluded that “the heterozygous state for fibrocystic disease of the pancreas could not be implicated as an important cause of chronic chest disease or of peptic ulceration”. These results agreed with those of Anderson et al. (Med J Aust 1962; 1:965).

These results may not apply today as chronic bronchitis was remarkably common in the general population then due to atmospheric pollution and tobacco smoking.

 

1968 Harries RL, Riley HD Jr. Cystic fibrosis in the American Indian. Pediatr 1968; 41:733-738.
These were the first two children with CF in the American Indian population. Both patients had convincingly high sweat tests and clinical features and were three years eight months and one year 11 months when reported. A sibling of case one who died had the typical pancreatic changes of cystic fibrosis. Caucasian ancestry, if present, was very distant and only on one side of the family and then three generations back. It was suggested that the rarity of CF in Mongolian and American Indians may be related to their common Asian ancestry before the American Indians crossed the Bering Straits into the Western Hemisphere. [PubMed]

 

1968 Bhakoo ON, Kumar R, Walia BN. Mucoviscidosis of lung. Report of a case. Indian J Pediatr 1968; 35: 183-185. [PubMed]

 

1968 Mehta S, Wadhwa UN, Mehta SK, Chhutani PN. Fibrocystic disease of the pancreas in India. Indian Pediatr 1968; 5:185-191. [PubMed]

First two reports of cystic fibrosis in India. Reviewed by Kabra SK et al (Cystic fibrosis in India. Pediatr Pulmonol 2007; 42:1087-1094.) [PubMed]

 

1974 Kulczycki LL, Schauf V. Cystic fibrosis in blacks in Washington DC. Am J Dis Child 1974; 127:64-67. [PubMed]
An intensive search for black people CF between 1962 to 1971 revealed 16 with cystic fibrosis. The clinical course was similar to the condition in whites. The calculated incidence was estimated as at least one in 17,033 black newborns. Later these findings were confirmed by others. More recently African mutations have been described by Michelle Ramsay and colleagues in South Africa (Carles et al, 1996 below).

 

1995 Gan K-H, Geus WP, Bakker W, Lamers CBHW, Heijerman HGM. Genetic and clinical features of patients with cystic fibrosis diagnosis after the age of 16 years. Thorax 1995; 50:1301-1304. [PubMed]

Predictably, adult patients with late diagnosis have a better prognosis. The differences between the early diagnosis of 4.6 years (ED) and late diagnosis 27.7 years (LD) were FEV1 52% vs 72.5%; Pseudomonas infection in 70% vs 24%; pancreatic insufficiency 81% vs 12%; homozygous for DF508 58% vs none.

In other series of adults with CF many have been diagnosed late and had other features suggesting many had milder mutations – supported by the high frequency of pancreatic sufficiency. However, it has been emphasised that these patients require just as careful follow up and treatment as those with the more usual clinical picture - rather than waiting until they develop more obvious chest symptoms (Lording A, et al. Pulmonary infection in mild variant cystic fibrosis: implications for care. J Cyst Fibros 2006; 5:101-104.[PubMed]).

 

1995 Gan K H, Veeze HJ, van den Ouweland AM, Halley DJ, Scheffer H, van der Hout, A, Overbeek SE, de Jongste JC, Bakker W, Heijerman HG. A cystic fibrosis mutation associated with mild lung disease. New Eng J Med 1995; 333:95-99. [PubMed]

Among Dutch patients A455E the second most common mutation and associated with preserved pancreatic function and some residual secretion of chloride across membranes. Thirty three patients with compound heterozygosity for the A455E mutation were compared with matched controls homozygous for the delta F508 mutation. In those with the A455E mutation diagnosis was later (mean age at diagnosis, 15.0 vs. 3.1 years; P < 0.001); fewer had pancreatic insufficiency (21.2 percent vs. 93.9 percent, P < 0.001), and none had diabetes mellitus (0 percent vs. 27.3 percent, P = 0.004). FEV1 and FVC were significantly higher (FEV1, 73.9 vs. 54.3 % predicted P = 0.002) and FVC, 88.7 vs. 76.3 % predicted P = 0.04). Fewer had chronic pseudomonas infection (33.3 vs. 60.6 % P = 0.02).

Although several mutations were known to be associated with less severe pancreatic disease, these findings showed a correlation between the A455E mutation and mild pulmonary disease resulting in a better prognosis than patients homozygous for the df508.

Dr Harry Heijerman (figure 1) was the Founding Editor of the Journal of Cystic Fibrosis. Under his guidance the journal was increasingly successful; the editorship was taken over by Prof. Gerd Döring in 2006 (see 2002 Journal of Cystic Fibrosis below). Gerd died in 2013 and the editorship passed to Scott Bell. Harry Heijerman is Physician at Haga Teaching Hospital, Deb Haag in the Netherlands and a leading figure in CF care and research in Europe.

 

Fig. 1: Harry Heijerman

 

1996 Carles S, Desgeorges M, Goldman A, Thiart R, Guittard C, Kitazos CA, de Ravel TJ, Westwood AT, Claustres M, Ramsay M. First report of CFTR mutations in black cystic fibrosis patients of southern African origin. J Med Genet 1996; 33:802-804. [PubMed]

Cystic fibrosis was thought to be rare in the black populations of Africa and only a few patients have been reported but they had not been studied at the molecular level. This report, from Michelle Ramsay’s laboratory, concerns the detection of CFTR mutations in three black South African patients. One was homozygous for the 3120 + 1G-->A mutation, while the other two were compound heterozygotes each with this mutation on one chromosome. The other mutations were G1249E and a previously unreported in frame 54 bp deletions within exon 17a involving nucleotides 3196-3249 (3196del54).

The 3120 + 1G-->A mutation was first described in black American patients and has been shown to be a relatively common mutation in this population (9-14% of CF chromosomes). It was also found in a black CF patient whose father, the 3120 + 1G-->A carrier, is from Cameroon. These data suggest that it is an old mutation which accounts for many of the CFTR mutations in black Africans (Also Pileggi A. 1962; Kulczycki LL et al, 1964 above and other reports find a low incidence in black patients).

 

 

1997 Loirat F, Hazout S, Lucotte G. G542X as a probable Phoenician cystic fibrosis mutation. Hum Biol 1997; 69:419-425. [PubMed]

When analyzed by origin, the frequency of the G542X CF mutation (the second most common CF mutation in Europe after DF508) varies between population groups in Europe being lower in north-eastern Europeans than in south-western Europeans. The more elevated values of G542X frequency correspond to ancient sites of occupation by occidental Phoenicians. N1303K as been linked to ancient Mediterranean populations and G551D associated with ancient Celtic tribes (Cashman SM et al, Hum Hered 1995; 45:6-12. [PubMed]).

 

2000 Kostuch M, Semczuk A, Szarewicz-Adamczyk W, Gasowska-Giszczak U, Wojcierowski J, Kulczycki L. Detection of CFTR gene mutations in patients suffering from chronic bronchitis. Arch Med Res 2000; 31: 97-100. [PubMed]
The purpose of this study from Poland was to search for CF gene mutations in patients suffering from chronic bronchitis. No less than five of 32 (16%) patients admitted to the Lublin School of Medicine, Poland between 1995 and 1996 with chronic bronchitis had CF gene mutations, all within the DeltaF508 region of the CFTR gene. All positive samples were obtained from patients heterozygous for the DeltaF508 mutation. The presence of the DeltaF508 mutation was considered statistically significant when the study group was compared to the study of Poland's general population. The results suggested an increased presence of the DeltaF508 mutation in Polish patients suffering from chronic bronchitis.

 

Earlier clinical studies using sweat tests from the UK did not show people with unrecognised or mild CF were present among patients considered to have chronic bronchitis (Muir D et al. 1962 above). Also in another study, perhaps more relevant to the present report, there was no increase in the frequency of chronic bronchitis among obligate CF heterozygotes (Batten et al, 1963 above). However, the prevalence of chronic bronchitis was very high in the UK general population at that time (presumably due to the high proportion of smokers and atmospheric pollution) and this may have obscured the influence of the CF carrier state. More recently complete mutational screening of 120 patients with chronic pulmonary disease detected mutations in 11/23 disseminated bronchiectasis, 7/25 emphysema, 5/27 chronic bronchitis, 5/26 lung cancer, 5/8 sarcoidosis (Bombieri et al, 1998. [PubMed]).  In a more recent study 17.74% of 31 patients with chronic obstructive pulmonary disease had a CFTR mutation (Divac et al, 2004). [PubMed] Although others searching for only the six common mutations found no increase in CFTR mutations in 100 patients with chronic bronchitis (Entzian P et al, 1995). [PubMed] However,  5/11 non-CF individuals who had allergic bronchopulmonary aspergillosis (ABPA) had one CFTR mutation (Miller PW et al, 1996. [PubMed]). Also CFTR function has been reported as significantly depressed in smokers (Cantin AM et al. Am J Resp Crit Care Med 2006; 173:1139-1144). [PubMed]

 

 

2000 Mickle JE, Cutting GR. Genotype-phenotype relationships in cystic fibrosis. Med Clin North Am 2000; 84:597-607. [PubMed]
The fact that variability is observed among people with the “classic” form of CF is evidence that other factors are important. The so-called “mild mutations” that allow partial function of CFTR are often associated with pancreatic sufficiency, occasionally identified with normal sweat gland function, and sporadically correlated with mild lung disease; also partially functioning mutants rarely prevent maldevelopment of the male reproductive tract; an exception is 3849 + 10 Kb C-->T.

These observations suggest that certain tissues require different levels of CFTR function to avoid the pathologic manifestations typical of CF. However; It is still true that, in the majority of patients, the outlook is more dependent on the standard of treatment the patient receives and whether that treatment is started before there is irreversible lung damage.

 

Fig. 4: Garry Cutting. From Johns Hopkins website.
Fig. 5. Manfred Ballman

From the Department of Pediatrics McKusick-Nathans Institute of Genetic Medicine. Johns Hopkins University School of Medicine, Baltimore. Prof. Garry Cutting (figure 4) is interested in the molecular biology and human genetics of ion channel disorders. Much of his current work is focused upon cystic fibrosis.

 

2000 Mekus F, Ballman M, Bronsveld I, Bijman J, Veeze H, Tummler B. Categories of DF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics. Twin Res 2000; 3:277-293. [PubMed]
A large European twin study searching for features for which the twins were discordant. Monozygotic twins with CF had less discordance than dizygotic twins indicating that, in part, CF severity is modulated by an inherited component in addition to the CFTR itself and the very important environmental factors.
Although there was increasing interest in the effect of modifying genes that affected the course of CF in affected individuals, in practical terms the influence of most was relatively small when compared with the influence of the standard of treatment the patient received.

 

Professor Ballman (figure 5) is based in the Paediatric Department and CF Centre in Hanover, Germany and involved in national and international CF matters.

 

2001 Iwasa S, Fujiwara M, Nagata M, Watanabe T. Three autopsied cases of cystic fibrosis in Japan. Pathol Internat 2001; 51:467-472. [PubMed]
The incidence of CF is very low in the Japanese. All three patients initially developed meconium ileus, and hepatobiliary and pancreatic changes became more severe as age increased. None had the DeltaF508 mutation. The authors reviewed 22 Japanese autopsied cases of CF in the literature. They suggested that the high incidence of meconium ileus in Japanese CF patients may relate to a clinically severe phenotype and reflect a different genetic background between Caucasians and Japanese. The incidence of CF in Japanese had been estimated previously at 1:350,000 considering 104 reported cases in 1997 (Yamashiro Y et al, 1997; 24:544-547. [PubMed]; also Nishimori I, Onishi S. Hereditary pancreatitis in Japan: a review of pancreatitis-associated gene mutations. Pancreatology 2001; 1:444-447. [PubMed]).

 

2001 Goldman A, Labrum R, Claustres M, Desgeorges M, Guittard C, Wallace A, Ramsay M. The molecular basis of cystic fibrosis in South Africa. Clin Genet 2001; 59:37-41. [PubMed]
The spectrum of CFTR mutations in three South African populations is presented. To date. a total of 192 white patients (384 chromosomes) with confirmed CF have been tested. deltaF508 accounts for 76% of the CF chromosomes in this group, with 3272-26A-->G, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. A further 11 mutations account for 6% of CF chromosomes. A total of 91% of the CF-causing mutations can now be detected in the South African white population. Haplotype analysis suggests a founder effect in South Africans of European origin for the two common CFTR mutations, 3272-26A-->G and 394delTT. The diagnosis of CF has been confirmed in 14 coloured and 12 black CF patients. In the coloured population, both the deltaF508 and 3120 + 1G-->A mutations occur at appreciable frequencies of 43 and 29%, respectively. In the black population, the most common CF-causing mutation, the 3120 + 1G-->A mutation, occurs at an estimated frequency of 46%. Four other mutations have been detected, resulting in the identification of a total of 62.5% of mutations in this population.

 

An interesting paper from Michelle Ramsay's department in South Africa documenting the mutations found in the coloured and black populations.

 

2002 Bobadilla JL, Macek M, Fine JP, Farrell PM. Cystic fibrosis: A worldwide analysis of CFTR mutations – correlation with incidence data and application to screening. Hum Mutat 2002; 19:575-606. [PubMed]
The results confirmed wide mutational heterogeneity throughout the world. They also examined CF incidence, DeltaF508 frequency, and regional mutational heterogeneity in a subset of populations and there is a significant positive correlation between DeltaF508 frequency and the CF incidence levels of regional populations. Regional analyses were also performed to search for trends in the distribution of CFTR mutations across migrant and related populations; this led to clarification of ancestry-genotype patterns that can be used to design CFTR multi-mutation panels for CF screening programs.

 

This is an immense study bringing together all the information from over 100 published papers to achieve a global understanding of the population molecular genetics associated with CF in an effort to increase understanding of ancestry-genotype relationships, to compare mutational arrays with incidence and to gain insight for decisions regarding screening program enhancement through CFTR mutational analysis.

 

2003 Li N, He B, Wang GF, Tang XY.  A case report of cystic fibrosis and review of 16 cases of cystic fibrosis in Chinese patients. [Chinese] Chinese Journal of Tuberculosis & Respiratory Diseases 2003; 26:559-562. [PubMed].

A 14-year-old Chinese girl presenting with recurrent productive cough since birth with parasinusitis and otitis media was confirmed to have CF in Peking University First Hospital. Chest CT scan showed bronchiectasis, more severe in the right upper lobe. Sweat tests were taken three times, and the values of Na(+) and Cl(-) were (126.6 +/- 5.4) mmol/L and (108.9 +/- 3.3) mmol/L, respectively. The examination of the pancreas showed no remarkable cystic changes on CT scan and there was no pancreatic insufficiency. Sixteen patients with CF in Chinese reported from 1974 to 1999 were reviewed. Sixteen of the 17 patients (7 males and 9 females, aged from 6 months to 25 years) had clinical data available for analysis. Eleven of them had died, nine before the age of 13. They all suffered from frequent episodes of pneumonia, while 14 had malnutrition and 4 had jaundice. The diagnostic procedures included clinical features, sweat test and autopsy. Four of them had DNA screened and four kinds of novel mutations in the cystic fibrosis transmembrane conductance regulator gene were found.

 

So Chinese patients with CF show similar clinical manifestations to patients in the European and North American populations, but the CFTR mutation was different. (See also Li N et al,2006 below).

 

2003 Frangolias DD, Ruan J, Wilcox PJ, Davidson AG, Wong LT, Berthiaume Y, Hennessey R, Freitag A, Pedder L, Corey M, Sweezey N, Zielenski J, Tullis E., Sandford AJ. Alpha 1-antitrypsin deficiency alleles in cystic fibrosis lung disease. Am J Resp Cell Mol 2003; 293):390-396.[PubMed]
The conclusions of this detailed study were that alpha1-AT genotype is not a major contributor to the variability of pulmonary disease severity in cystic fibrosis.

 

2005 Drumm ML, Konstan MW, Schluchter MD, Handler A, Pace R, Zou F, Zariwala M, Fargo D, Xu A, Dunn JM, Darrah RJ, Dorfman R, Sandford AJ, Corey M, Zielenski J, Durie P, Goddard K, Yankaskas JR, Wright FA, Knowles MR. Gene Modifier Study Group. Genetic modifiers of lung disease in cystic fibrosis. N Eng J Med 2005; 353:1443-1453. [PubMed]
Polymorphisms in genes other than the CFTR gene may modify the severity of pulmonary disease in patients with cystic fibrosis. 808 patients who were homozygous for the DeltaF508 mutation had either severe or mild lung disease (i.e. they were in the lowest or highest quartiles for FEV1). The authors genotyped 16 polymorphisms in 10 genes that had been reported by others as modifiers of disease severity in CF and tested for an association in the patients with severe disease (263 patients) or mild disease (545). In a second study, of 498 patients, with various CFTR genotypes and a range of FEV1 values, they tested for an association of the TGFbeta1 codon 10 CC genotype with low FEV1.

In the first study, significant allelic and genotypic associations with phenotype were seen only for TGFbeta1 (the gene encoding transforming growth factor beta1). The second study confirmed the association of the TGFbeta1 codon 10 CC genotype with more severe lung disease.

 

2006 McKone EF, Goss CH, Aitkin ML. CFTR genotype as a predictor of prognosis in cystic fibrosis. Chest 2006; 130:1441-1447. [PubMed]
Data from CF Foundation registry on 15,651 patients was analysed and showed that the “high risk” CFTR genotype carried a twofold increased risk of death compared to those with “low risk” genotype. Of patients who died, high risk median age of death 24.2 years and low risk 37.6 years. Most reassurance can be provided for patients in the low risk group although there is substantial phenotypic variability.

 

The outlook for people with CF is mainly determined by the treatment they receive although a small number certainly do have mutations which are associated with milder disease, often associated with pancreatic sufficiency and a much later presentation and diagnosis.

 

2006 Li N, Pei P, Bu DF, He B, Wang GF. A novel CFTR mutation found in a Chinese patient with cystic fibrosis. Chin Med J 2006; 119:103-109. [PubMed]
A Chinese girl of 16 years old was diagnosed as having CF at the age of 14 years. A heterozygous novel missense mutation of 699 C --> A, which results in the amino acid change of N189K, was identified in exon 5. In addition, a heterozygous 3821 - 3823 delT mutation in exon 19 was found in CFTR.

CF is very rare on Chinese people; only twenty were reported from 1974 to 2004 were also reviewed in this paper. DelF508 mutation was not found in any of the nine cases whose CFTR mutations were analyzed and suggests that the genotype of Chinese CF may be different from those in Europe and N America.

 

2006 Ziedalski TM, Kao PN, Henig NR, Jacobs SS, Ruoss SJ. Prospective analysis of cystic fibrosis transmembrane regulator mutations in adults with bronchiectasis or pulmonary nontuberculous mycobacterial infection. Chest 2006; 130:995-1002.[PubMed]
Fifty adult patients at Stanford University Medical Center with a diagnosis of bronchiectasis and/or pulmonary NTM infection were prospectively characterized by sweat chloride measurement, comprehensive mutational analysis of CFTR, and sputum culture results.

A new diagnosis of cystic fibrosis (CF) was established in 10 patients (20%). Sweat chloride concentration was elevated > 60 mEq/dL (diagnostic of CF) in seven patients (14%), and from 40 to 60 mEq/dL in eight patients (16%). The frequency of CFTR mutations was elevated above that expected in the general population: heterozygous DeltaF508 (12% vs 3%), R75Q (14% vs 1%), and intron 8 5T (17% vs 5 to 10%).

 

 

 

2008 Castellani C, Cuppens H, Macek M Jr, Cassiman JJ, Kerem E, Durie P, Tullis E, Assael BM, Bombieri C, Brown A, Casals T, Claustres M, Cutting GR, Dequeker E, Dodge J, Doull I, Farrell P, Ferec C, Girodon E, Johannesson M, Kerem B, Knowles M, Munck A, Pignatti PF, Radojkovic D, Rizzotti P, Schwarz M, Stuhrmann M, Tzetis M, Zielenski J, Elborn JS. Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice. J Cyst Fibros 2008; 7:179-196. [PubMed]
It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. This is a major review by an international group of experts of the best way to use mutation analysis in cystic fibrosis.

 

 

 

2008 Douros K. Loukou I. Doudounakis S. Tzetis M. Priftis KN. Kanavakis E. Asthma and pulmonary function abnormalities in heterozygotes for cystic fibrosis transmembrane regulator gene mutations. Int j clin exp med 2008; 1:345-349. [PubMed]                                               The aim of this study is to evaluate the association between CFTR gene mutations with asthma and pulmonary function abnormalities. For this purpose, 214 mutation carriers were compared to 185 non-carriers. Although the relative risk of asthma did not differ between groups (OR=0.61, 95% CI: 0.23-1.61, p=0.32), the values of FEV1, and FEV1/FVC ratio were lower in carriers (p=0.001, and p<0.001, respectively).

The authors suggested that this may imply that heterozygosity may be related with a silent obstructive pulmonary profile.

 

2009 Dequeker E, Stuhrmann M, Morris MA, Casals T, Castellani C, Claustres M, et al. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders – updated European recommendations. Eur J Hun Genet 2009; 17:51-65.

An increasing number of laboratories offering molecular genetic analysis of the CFTR gene and the growing use of commercial kits strengthen the need for an update of previous best practice guidelines (published in 2000). The importance of organizing regional or national laboratory networks, to provide both primary and comprehensive CFTR mutation screening, is stressed. Current guidelines focus on strategies for dealing with increasingly complex situations of CFTR testing. Diagnostic flow charts now include testing in CFTR-related disorders and in fetal bowel anomalies. Emphasis is also placed on the need to consider ethnic or geographic origins of patients and individuals, on basic principles of risk calculation and on the importance of providing accurate laboratory reports. Finally, classification of CFTR mutations is reviewed, with regard to their relevance to pathogenicity and to genetic counselling.

 

2009 Bartlett JR, Friedman KJ, Ling SC, et al. Gene Modifier Study Group. Genetic modifiers of liver disease in cystic fibrosis. JAMA 2009; 302:1076-1083. [PubMed]
A subset (approximately 3%-5%) of patients with CF develops severe liver disease with portal hypertension. The objective of the study was to assess whether any of 9 polymorphisms in 5 candidate genes (alpha(1)-antitrypsin or alpha(1)-antiprotease [SERPINA1], angiotensin-converting enzyme [ACE], glutathione S-transferase [GSTP1], mannose-binding lectin 2 [MBL2], and transforming growth factor beta1 [TGFB1]) are associated with severe liver disease in patients with CF.

The authors concluded that the SERPINA1 Z allele is a risk factor for liver disease in CF. Patients who carry the Z allele are at greater risk (OR, approximately 5) of developing severe liver disease with portal hypertension.

 

2009 Thauvin-Robinet C. Munck A. Huet F. Génin E. Bellis G. Gautier E. et al. Collaborating Working Group on R117H.The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening. J Med Genet 2009; 46:752-758. [PubMed]
Phenotypic variability associated with certain mutations makes genetic counselling difficult, notably for R117H, whose disease phenotype varies from asymptomatic to classical CF. The high frequency of R117H observed in CF newborn screening has also introduced diagnostic dilemmas. The aim of this study was to evaluate the disease penetrance for R117H in order to improve clinical practice.

The phenotypes in all individuals identified in France as compound heterozygous for R117H and F508del, the most frequent CF mutation, were described. The allelic prevalences of R117H (p(R117H)), on either intron 8 T5 or T7 background, and F508del (p(F508del)) were determined in the French population, to permit an evaluation of the penetrance of CF for the [R117H]+[F508del] genotype. Clinical details were documented for 184 [R117H]+[F508del] individuals, including 72 newborns.

The disease phenotype was predominantly mild; one child had classical CF, and three adults' severe pulmonary symptoms. In 5245 healthy adults, p(F508del) was 1.06%, p(R117H;T7) 0.27% and p(R117H;T5)<0.01%. The theoretical number of [R117H;T7]+[F508del] individuals in the French population was estimated at 3650, whereas only 112 were known with CF related symptoms (3.1%). The penetrance of classical CF for [R117H;T7]+[F508del] was estimated at 0.03% and that of severe CF in adulthood at 0.06%.

 

The authors suggest that these results suggest that R117H should be withdrawn from CF mutation panels used for screening programmes. They suggest real impact of so-called disease mutations should be assessed before including them in newborn or preconceptional carrier screening programmes.

 

2010 Spadafora D, Hawkins EC, Murphy KE, Clark LA, Ballard ST. Naturally occurring mutations in the canine CFTR gene. Physiol Genom 2010; 42:480-485. [PubMed]
Naturally occurring cystic fibrosis (CF)-causing mutations in the CFTR gene have not been identified in any non-human animal species. Since domestic dogs are known to develop medical conditions associated with atypical CF in humans (e. g. , bronchiectasis and pancreatitis), the authors hypothesized that dogs with these disorders are likely have a higher expression rate of CFTR mutations than the at-large population. Temporal temperature-gradient gel electrophoresis (TTGE) was used to screen canine CFTR in 400 animals: 203 dogs diagnosed with pancreatitis, 23 dogs diagnosed with bronchiectasis, and 174 dogs admitted to clinics for any illness ("at-large" dogs).

Twenty-eight dogs were identified with one of four CFTR missense mutations. P1281T and P1464H mutations occur in relatively unconserved residues. R1456W is analogous to the human R1453W mutation, which has approximately 20% of normal CFTR function and is associated with pancreatitis and panbronchiolitis. R812W disrupts a highly conserved protein kinase A recognition site within the regulatory domain.

The authors conclude that naturally occurring CFTR mutations are relatively common in domestic dogs and can be detected with TTGE. No substantive differences in mutation frequency were observed between the at-large, pancreatitis, and bronchiectasis dogs.

 

This is of interest and CFTR mutations in dogs is a new but not particularly practical relevant finding. Impressive to collect so many "patients". with these various disorders.

 

2011 Wright FA, Strug LJ, Doshi VK, Commander CW, Blackman SM, Sun L, Berthiaume Y, Cutler D, Cojocaru A, Collaco JM, Corey M, Dorfman R, Goddard K, Green D, Kent JW Jr, Lange EM, Lee S, Li W, Luo J, Mayhew GM, Naughton KM, Pace RG, Pare P, Rommens JM, Sandford A, Stonebraker JR, Sun W, Taylor C, Vanscoy LL, Zou F, Blangero J, Zielenski J, O'Neal WK, Drumm ML, Durie PR, Knowles MR, Cutting GR. Genome-wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2. Nat Genet 2011; 43:539-546. [PubMed]
A combined genome-wide association and linkage study was used to identify loci causing variation in cystic fibrosis lung disease severity. The authors identified a significant association (P = 3.34 x 10(-8)) near EHF and APIP (chr11p13) in p.Phe508del homozygotes (n = 1,978). The association replicated in p.Phe508del homozygotes (P = 0.006) from a separate family based study (n = 557), with P = 1.49 x 10(-9) for the three-study joint meta-analysis. Linkage analysis of 486 sibling pairs from the family based study identified a significant quantitative trait locus on chromosome 20q13.2 (log(10) odds = 5.03). These findings provide insight into the causes of variation in lung disease severity in cystic fibrosis and suggest new therapeutic targets for this life-limiting disorder.

 

A number of studies have searched for genetic modifiers of the severity of CF. This is one such study although it is not clear how this knowledge will be used as a therapeutic target unless by modifying the locus described. Undoubtedly  in 2014 the standard of treatment received during childhood and subsequently seems to be the most important factor in determining the patient's condition and ultimate survival.

 

2013 Tsui LC. Dorfman R. The cystic fibrosis gene: a molecular genetic perspective. [Review]

Cold Spring Harbor Perspectives in Medicine 2013; 3(2):a009472. Free Download available[PubMed]

The positional cloning of the gene responsible for cystic fibrosis (CF) was the important first step in understanding the basic defect and pathophysiology of the disease. This study aims to provide a historical account of key developments as well as factors that contributed to the cystic fibrosis transmembrane conductance regulator (CFTR) gene identification work.

A redefined gene structure based on the full sequence of the gene derived from the Human Genome Project is presented, along with brief reviews of the transcription regulatory sequences for the CFTR gene, the role of mRNA splicing in gene regulation and CF disease, and, various related sequences in the human genome and other species. Because CF mutations and genotype-phenotype correlations are covered by our colleagues (Ferec C, Cutting GR. 2012. Assessing the disease-liability of mutations in CFTR. Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a009480), the authors only attempt to provide an introduction of the CF mutation database here for reference purposes.

 

 

 

2011 Keiser NW. Engelhardt JF. New animal models of cystic fibrosis: what are they teaching us? Curr Opin Pulmon Med 2011; 17:478-483. [PubMed]

Cystic fibrosis is the first human genetic disease to benefit from the directed engineering of three different species of animal models (mice, pigs, and ferrets). Recent studies on the cystic fibrosis pig and ferret models are providing new information about the pathophysiology of cystic fibrosis in various organ systems. Additionally, new conditional cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice are teaching unexpected lessons about CFTR function in surprising cellular locations. Comparisons between these animal models and the human condition are key to dissecting the complexities of disease pathophysiology in cystic fibrosis.

Recently cystic fibrosis pigs and ferrets have provided new models to study the spontaneous development of disease in the lung and pancreas, two organs that are largely spared overt spontaneous disease in cystic fibrosis mice.

New cystic fibrosis mouse models are now interrogating CFTR functions involved in growth and inflammation at an organ-based level using conditional knockout technology. Together, these models are providing new insights on the human condition.

Basic and clinical cystic fibrosis research will benefit greatly from the comparative pathophysiology of cystic fibrosis mice, pigs, and ferrets. Both similarities and differences between these three cystic fibrosis models will inform pathophysiologically important mechanisms of CFTR function in humans and aid in the development of both organ-specific and general therapies for cystic fibrosis.

This is a very useful summary of the role of animal models the literature surrounding which is increasing at a pace!

 

 

 

1992 Snouweart JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, et al. An animal model for cystic fibrosis made by gene targeting. Science 1992; 257:1083-1088. (August 1992). [PubMed]
The authors generated a mouse line in which the cystic fibrosis transmembrane conductance regulator (CFTR) gene was mutated by gene targeting. Like patients with CF, mice lacking a functional CFTR gene (CFTR (-/-) mice) showed increased numbers of goblet cells and obstruction of glands with inspissated eosinophilic secretions. However, the most severe pathological changes in these mice were confined to the intestinal tract and gallbladder; there were only minor pathological alterations in the lungs and upper airways of the CFTR (-/-) mice. Possible explanations for the apparent lack of respiratory disease are the young age at which the animals were examined and the pathogen-free environment in which they were housed i.e. there had not been time to develop respiratory problems.

The production of a “CF Mouse” was a major advance. Oliver Smithies was awarded a share of the Nobel Prize in 2007 for this work. The award was shared with Professor Martin Evans (Ratcliff et al, 1993 below). It is worth recalling that the lungs of CF infants who die in the newborn period also show little in the way of histological abnormality.

 

Fig 1: Julia Dorin. From www.hgu.mrc.ac.uk website

Fig 2: Sir Martin Evans. From Cardiff University School of Biosciences website.

1992 Dorin JR, Dickinson P, Alton EWFW, Smith SN, Geddes DM, Stevenson BJ, et al. Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 1992; 359:211-215. (Sept 1992). [PubMed]
To make this mouse the cystic fibrosis transmembrane conductance regulator gene was disrupted in embryonal stem cells using an insertional gene targeting vector. Germ-line chimaeras were derived and the offspring of heterozygous crosses studied. These homozygous mutant mice survived beyond weaning and in vivo electro physiology demonstrates the predicted defect in chloride ion transport and can distinguish between each genotype. Also histological analysis showed important hallmarks of human disease pathology, including abnormalities of the colon, lung and vas deferens.

Julia Dorin's (figure 1) was one of the three groups to create a CF mouse model - the "Edinburgh mouse", created by insertional mouse mutation. It provided a valid model system for the development and testing of therapies for cystic fibrosis patients and was a major advance

 

1993 Ratcliff R, Evans MJ, Cuthbert AW, MacVinish LJ, Foster D, Anderson JR, et al. Production of a severe cystic fibrosis mutation in mice by gene targeting. Nature Genet 1993; 4:35-41. [PubMed]
The authors used gene targeting in embryonic stem cells to introduce an HPRT mini-gene into the coding sequence of the murine cystic fibrosis gene (cftr). This insertion introduces a termination codon in frame with the cftr coding sequence to terminate prematurely the CFTR protein within the first nucleotide binding domain. Animals homozygous for the cftr disruption fail to thrive and display a range of symptoms including meconium ileus, distal intestinal obstructions, gastrointestinal mucus accumulation and blockage of pancreatic ducts. The animals also show lacrimal gland pathology. Tracheal and caecal transepithelial current measurements demonstrate the lack of a cAMP and can be used to study gene therapy strategies.

This and the two preceding papers (Snouweart JN et al, 1992 above; Dorin et al, 1992 above) describing the creation of three types of CF mice were an essential development for further research into correction of the basic genetic defect.


Sir Martin Evans (figure 2) first of Cambridge and then Cardiff, was awarded a share of the Nobel Prize n 2007 for his work on knockout mice – he shared the prize with Mario Capechi of Utah and Oliver Smithies of North Carolina. The Nobel citation describes the trio’s discovery for introducing specific gene modifications in mice by the use of embryonic stem cells. Martin Evans was the first to grow stem cells from early mouse embryos. He injected stem cells from one strain into the embryos of another strain then implanted the embryo into a surrogate mother leading to mosaic-celled mice.

 

2010 Mall MA, Button B, Johanness on B, Zhou Z, Levirate A, Caldwell RA, Sherbet SC, Schultz C, O'Neal WK, Preordained S, Homelier E, Rosier BC, Grubb BR, Boucher RC. Airway surface liquid volume regulation determines different airway phenotypes in Liddle compared with betaENaC-overexpressing mice. J Biolog Chem 2010; 285:26945-55. [PubMed]
Studies in CF patients and mice overexpressing the epithelial Na(+) channel beta-subunit (betaENaC-Tg) suggest that raised airway Na(+) transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function betaENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, betaENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na(+) transport measured in Ussing chambers ("flooded" conditions) was raised in both Liddle and betaENaC-Tg mice. Because enhanced Na(+) transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic "thin film" conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na(+) absorption were intact in Liddle but defective in betaENaC-Tg mice.

The authors conclude that the capacity to regulate Na(+) transport and ASL volume, not absolute Na(+) transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.

 

2010 Zaman MM, Martin CR, Andersson C, Bhutta AQ, Cluette-Brown JE, Laposata M, Freedman SD. Linoleic acid supplementation results in increased arachidonic acid and eicosanoid production in CF airway cells and in cftr-/- transgenic mice. Am J Physiol - Lung C 2010; 299:L599-606. [PubMed]

Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. The authors hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidonate-derived pro inflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, they determined the effect of linoleic acid supplementation on downstream pro inflammatory biomarkers in two CF models: 1) in vitro cell culture model using 16HBE14o(-) sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using cftr(-/-) transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, PGE(2), and PGF(2alpha) secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation. cftr(-/-) Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and pro inflammatory mediators.

 

These findings are important as there was considerable interest in linoleic acid supplementation in people with CF - even a trial of intravenous linoleic acid in the Seventies. The situation regarding essential fatty acids remain unclear to this writer! (See also Topics -> Gastroenterology -> Fatty acids).

 

Mall MA. Graeber SY. Stahl M. Zhou-Suckow Z. Early cystic fibrosis lung disease: Role of airway surface dehydration and lessons from preventive rehydration therapies in mice. Int J Biochem Cell Biol 2014; 52:174-9. 24561284=[PubMed]
Airway surface dehydration is a key disease mechanism in CF, however, its role in the in vivo pathogenesis and as therapeutic target in early lung disease remains poorly understood. Mice with airway-specific over expression of the epithelial Na(+) channel (betaENaC-Tg) recapitulate airway surface dehydration and phenocopy CF lung disease. Recent studies in neonatal betaENaC-Tg mice demonstrated that airway surface dehydration produces early mucus plugging in the absence of mucus hyper secretion, which triggers airway inflammation, promotes bacterial infection and causes early mortality. Preventive rehydration therapy with hypertonic saline or amiloride effectively reduced mucus plugging and mortality in neonatal betaENaC-Tg mice.
These results support clinical testing of preventive/early rehydration strategies in infants and young children with CF.
 
This study provides further evidence of the basic importance of dehydration within the airways. Interesting that amiloride “effectively reduced mucus plugging” in view of its disappointing performance in previous clinical trials

 

Morro M. Teichenne J. Jimenez V. Kratzer R. Marletta S. Maggioni L. Mallol C. Ruberte J. Kochanek S. Bosch F. Ayuso E. Pancreatic transduction by helper-dependent adenoviral vectors via intraductal delivery. Gene Ther 2014; 25:824-36. [PubMed]25046147+
Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, the authors generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days post-injection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days post-injection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery.
The authors suggest that these data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue.
 
Although most attention regarding gene therapy in CF has been devoted to treatment of the lungs, the long term effects of pancreatic damage, in particular the diabetes mellitus, are of increasing importance as survival increases. In this respect it is encouraging that the new mutation specific drug ivacaftor is reported to have a favourable effect on insulin secretion in people with cystic fibrosis and the GF551D mutation (Bellin MD et al. Insulin secretion improves in cystic fibrosis following ivacaftor correction of CFTR: a small pilot study. Pediatr Diabetes. 2013;14:417-21. [PubMed] Free article available).

 

 

 

 

2008 Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyansky J, McCray PB Jr, Zabner J, Prather RS, Welsh MJ. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 2008; 26; 321(5897):1837-1841. [PubMed]
Pigs share many anatomical and physiological features with humans. The authors generated pigs with a targeted disruption of both CFTR alleles that developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF.
Progress was reviewed by Michael Welsh and colleagues in 2009 (Welsh et al, 2009. [PubMed]); also some features of the pathology of the CF pig were reviewed. Lesions resembling those in humans with CF were detected in intestine, pancreas, liver, gallbladder, and cystic duct. These organs had four common features. First, disease was accelerated compared with that in humans, which could provide a strategy to discover modifying factors. Second, affected organs showed variable hyperplastic, metaplastic, and connective tissue changes, indicating that remodeling was a dynamic component of fetal life. Third, cellular inflammation was often mild to moderate and not always present, which raises new questions as to the role of cellular inflammation in early disease pathogenesis. Fourth, epithelial mucus-producing cells were often increased, producing a striking accumulation of mucus with a layered appearance and resilient structure. Thus, mucus cell hyperplasia and mucus accumulation appear to play prominent roles in early disease. It was shown that luboprostone stimulates secretion from the tracheal submucosal glands of sheep, pigs and humans (Joo N S et al. 2009.[PubMed]). Subsequently hyposecretion of fluid from submucosal glands of CFTR-deficient pigs was demonstrated (Joo N S et al. 2010. [PubMed]).
[There is a free detailed and fully illustrated PMC article entered via Pubmed]


 

2009 Welsh MJ. Rogers CS. Stoltz DA. Meyerholz DK. Prather RS. Development of a porcine model of cystic fibrosis. Trans Am Clin Climat Assoc 2009; 120:149-162. [PubMed]

Because of the similarities between human and swine anatomy, biochemistry, physiology, size, and genetics, the authors chose to develop a porcine model of CF. They used homologous recombination in primary cultures of porcine fibroblasts to disrupt the CFTR gene and then used those cells as nuclear donors for somatic cell nuclear transfer. After crossing heterozygous pigs, they produced CFTR-/- pigs. The newborn CFTR null piglets manifested meconium ileus, pancreatic destruction, early focal biliary cirrhosis, and gall bladder abnormalities that were very similar to those observed in humans with CF. At birth, there were no abnormalities in the airway epithelium or submucosal glands and no evidence of inflammation, consistent with findings in the newborn human.

They hope that this porcine model will help elucidate the pathogenesis of CF and thereby lead to the development of new mechanism-based therapies.

 

2010 Chen JH, Stoltz DA, Karp PH, Ernst SE, Pezzulo AA, Moninger TO, Rector MV, Reznikov LR, Launspach JL, Chaloner K, Zabner J, Welsh MJ. Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia. Cell 2010; 143:911-923. [PubMed]

Defective transepithelial electrolyte transport is thought to initiate CF lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR-(/)- pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. The authors studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR-(/)- epithelia showed markedly reduced Cl- and HCO3- transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR-(/)- pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl- conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl- and HCO3- in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.

 

Further interesting findings from the recently created CF pig, questioning the long held belief that sodium hyperabsorption occurs in addition to defective chloride and bicarbonate transport in the CF epithelium. However, there are many previous publications indicating a reduced periciliary liquid depth which interferes with mucociliary clearance from the mucosa even though this was not confirmed in this present study in CF pigs.

 

2010 Meyerholz DK, Stoltz DA, Namati E, Ramachandran S, Pezzulo AA, Smith AR, Rector MV, Suter MJ, Kao S, McLennan G, Tearney GJ, Zabner J, McCray PB Jr, Welsh MJ. Loss of cystic fibrosis transmembrane conductance regulator function produces abnormalities in tracheal development in neonatal pigs and young children. Am J Resp Crit Care 2010; 182:1251-1261.

[PubMed]

Detailed studies of the trachea of CF pigs and young children with CF revealed three abnormalities in the pigs. The trachea and main bronchi were narrow, the trachea was less circular than controls, the tracheal smooth muscle was abnormal and submucosal glands were hypoplastic. A less circular trachea was also present in scans from children with CF less than 2 years old and there was a reduced tracheal lumen in neonates with CF. So these changes appear to begin in fetal life.

 

This finding would explain the occasional infant with CF who seems to have a difficult chronic tight wheezy cough as occurs in infants with tracheomalacia and whose symptoms are not amenable to treatment with bronchodilators, antibiotics or steroids but gradually improve with time as the infant grows and the airways increase in size.

 

2010 Li H, Ganta S, Fong P. Altered ion transport by thyroid epithelia from CFTR(-/-) pigs suggests mechanisms for hypothyroidism in cystic fibrosis. Exper Physiol 2010; 95:1132-1144

[PubMed]

Subclinical hypothyroidism has been linked to cystic fibrosis, and the cystic fibrosis transmembrane conductance regulator (CFTR) shown to be expressed in the thyroid. The thyroid epithelium secretes Cl- and absorbs Na(+) in response to cAMP. These studies definitively demonstrate that CFTR mediates cAMP-stimulated Cl- secretion in a well-differentiated thyroid culture model and that knockout of CFTR promotes increased Na(+) absorption by a mechanism other than increased ENaC expression. These findings suggest several models for the mechanism of cystic fibrosis-associated hypothyroidism.

Suggest read the full abstract for details. There have been a number of publications on the relationship between CF and hypothyroidism in the older literature. (Vagenakis, A G et al, Med Clin N Am 1975; 59:1075-1088.). The use of iodides to reduce sputum viscosity was common practice in the past and is mentioned by a number of authors including Harry Shwachman who mentioned their use as helpful in a number of his review articles. In a subsequent report from Shwachman and colleagues (Segall-Blank, M et al, Thyroid gland function and pituitary TSH reserve in patients with cystic fibrosis. J Pediatr 1981; 98:218-222.[PubMed]) they state that, in view of the reported enhanced sensitivity to iodide-induced hypothyroidism in patients with cystic fibrosis, studies were carried out to determine the possible mechanism of this abnormality. But their findings did not delineate the mechanism whereby patients with CF develop iodide-induced hypothyroidism (also see Ruben et al, 1960 above; Dolan & Gibson, 1971 above. Also Topic -> Endocrine -> Thyroid function). The present studies reported here seem to throw some new light on the subject.

 

2010 Joo NS, Cho HJ, Khansaheb M, Wine JJ. Hyposecretion of fluid from tracheal submucosal glands of CFTR-deficient pigs. J Clin Invest 2010; 120:3161- 6.[PubMed]

The recent production of pigs lacking CFTR and pigs expressing the most common CF-causing CFTR mutant, DeltaF508, provide another model that might help clarify the pathophysiology of CF airway disease. Here, the authors studied individual submucosal glands from 1-day-old piglets in situ in explanted tracheas, using optical methods to monitor mucus secretion rates from multiple glands in parallel. Secretion rates from control piglets (WT and CFTR+/-) and piglets with CF-like disease (CFTR-/- and CFTR-/DeltaF508) were measured under 5 conditions: unstimulated (to determine basal secretion), stimulated with forskolin, stimulated with carbachol, stimulated with substance P, and, as a test for synergy, stimulated with forskolin and a low concentration of carbachol. Glands from piglets with CF-like disease responded qualitatively to all agonists like glands from human patients with CF, producing virtually no fluid in response to stimulation with forskolin and substantially less in response to all other agonists except carbachol. These data are a step toward determining whether gland secretory defects contribute to CF airway disease.

Yet another of the interesting study involving the recently available CF pigs from Jeffrey Wine's team confirming the similarity with human CF tracheal submucosal glands.

 

2010 Sun X, Sui H, Fisher JT, Yan Z, Liu X, Cho HJ, Joo NS, Zhang Y, Zhou W, Yi Y, Kinyon JM, Lei-Butters DC, Griffin MA, Naumann P, Luo M, Ascher J, Wang K, Frana T, Wine JJ, Meyerholz DK, Engelhardt JF. Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis. J Clin Invest 2010; 120:3149-60.[PubMed]

Here, the authors report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, the authors cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Some details of the recently available CF ferret. The ability to clone gut-corrected transgenic CFTR-knockout kits that expressed the species appropriate CFTR in the intestine was very interesting.

 

2010 Stoltz DA, Meyerholz DK, Pezzulo AA, Ramachandran S, Rogan MP, Davis GJ, Hanfland RA, Wohlford-Lenane C, Dohrn CL, Nelson GA 4th, Chang EH, Taft PJ, Ludwig PS, Estin M, Hornick EE, Launspach JL, Samuel M, Rokhlina T, Karp PH, Ostedgaard LS, Uc A, Starner TD, Horswill AR, Brogden KA, Prather RS, Richter SS, Shilyansky J, McCray PB Jr, Zabner J, Welsh MJ. Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth. Sci Transl Med 2010; 2:29ra31.[PubMed]

Within months of birth, CF pigs spontaneously developed hallmark features of CF lung disease, including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting that the lungs of CF pigs have a host defense defect against a wide spectrum of bacteria. In humans, the temporal and causal relations between inflammation and infection have remained uncertain. To investigate these processes, the authors studied newborn pigs. Their lungs showed no inflammation but were less often sterile than controls. Moreover, after introduction of bacteria into their lungs, pigs with CF failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. The finding that pigs with CF have a host defense defect against bacteria within hours of birth provides an opportunity to further investigate CF pathogenesis and to test therapeutic and preventive strategies that could be deployed before secondary consequences develop.

Description of some of the features of the CF pig - a major advance in the study of CF already increasing understanding of human disease. Initial findings suggest that defective bacterial elimination precedes inflammation - although it does seem as if there are reasons for excessive inflammation when it does occur.

 

2010 Wine JJ. The development of lung disease in cystic fibrosis pigs. Sci Transl Med 2010; 2:29ps20. [PubMed]

The leading cause of death in people with CF is lung disease; yet despite enormous research efforts, its pathogenesis is not well understood. Because CF mice do not develop human-like airway disease, CF pigs were produced on the premise that they would offer insights into the pathogenesis of CF lung disease. Unfortunately CF pigs develop severe intestinal blockage and often die shortly after birth. Now, a team of scientists report results from five CF pigs that lived for 2 to 6 months; during this time, they developed some of the key features of human CF lung disease and revealed that infection precedes inflammation.

The discussion as to whether infection precedes inflammation or whether the inflammation is the first abnormality has been a source of discussion for many years. It seems that infection precedes inflammation as suggested here but that when inflammation occurs it may be excessive and prolonged.

 

2010 Meyerholz DK, Stoltz DA, Pezzulo AA, Welsh MJ. Pathology of gastrointestinal organs in a porcine model of cystic fibrosis. Am J Pathol 2010; 176:1377-89. [PubMed]

To better understand the initial stages of CF, the authors studied the gastrointestinal pathology of CFTR-/- pigs. By studying newborns, they avoided secondary changes attributable to environmental interactions, infection, or disease progression. Lesions resembling those in humans with CF were detected in intestine, pancreas, liver, gallbladder, and cystic duct. These organs had four common features. First, disease was accelerated compared with that in humans, which could provide a strategy to discover modifying factors. Second, affected organs showed variable hyperplastic, metaplastic, and connective tissue changes, indicating that remodeling was a dynamic component of fetal life. Third, cellular inflammation was often mild to moderate and not always present, which raises new questions as to the role of cellular inflammation in early disease pathogenesis. Fourth, epithelial mucus-producing cells were often increased, producing a striking accumulation of mucus with a layered appearance and resilient structure.

Thus, mucus cell hyperplasia and mucus accumulation play prominent roles in early disease. These findings also have implications for CF lung disease, and they lay the foundation for a better understanding of CF pathogenesis.

This is a very interesting report of the early changes in the gastrointestinal organs of CF pigs. Just as the CF mice were a major advance in 1992/3 so the porcine CF model is proving of immense value.

 

2010 Wine JJ. The development of lung disease in cystic fibrosis pigs. Science Translational Medicine 2010; 2:29ps20. [PubMed]
The leading cause of death in cystic fibrosis (CF) patients is lung disease; yet despite enormous research efforts, its pathogenesis is not well understood. Because CF mice do not develop human-like airway disease, CF pigs were produced on the premise that they would offer insights into the pathogenesis of CF lung disease. Unfortunately CF pigs develop severe intestinal blockage and often die shortly after birth. Now, a team of scientists report results from five CF pigs that lived for 2 to 6 months; during this time, they developed some of the key features of human CF lung disease and revealed that infection precedes inflammation.

The discussion as to whether infection precedes inflammation or whether the inflammation is the first abnormality has been a source of discussion for many years. It seems that infection precedes inflammation as suggested here but that when inflammation occurs it may be excessive and prolonged.

 

2010 Rogan MP, Reznikov LR, Pezzulo AA, Gansemer ND, Samuel M, Prather RS, Zabner J, Fredericks DC, McCray PB Jr, Welsh MJ, Stoltz DA. Pigs and humans with cystic fibrosis have reduced insulin-like growth factor 1 (IGF1) levels at birth. Proc Nat Acad Sci 2010; 107:20571-5. [PubMed]
People with cystic fibrosis (CF) exhibit growth defects. That observation has been attributed, in part, to decreased insulin-like growth factor 1 (IGF1) levels, and the reduction has been attributed to malnutrition and pulmonary inflammation. However, patients with CF already have a reduced weight at birth, a manifestation not likely to be secondary to poor intrauterine nutrition or inflammation. Also CF pigs were smaller than their non-CF littermates and had lower IGF1 levels. To better understand the basis of IGF1 reduction, the authors studied newborn CF pigs and found low IGF1 levels within 12 hours of birth. Moreover, humerus length and bone mineral content were decreased, consistent with less IGF1 activity in utero. These findings led them to test newborn humans with CF, and they found that they also had reduced IGF1 levels. Discovering lower IGF1 levels in newborn pigs and humans indicates that the decrease is not solely a consequence of malnutrition or pulmonary inflammation and that loss of cystic fibrosis transmembrane conductance regulator function has a more direct effect. Consistent with this hypothesis, they discovered reduced growth hormone release in organotypic pituitary slice cultures of newborn CF pigs. These findings may explain the long-standing observation that CF newborns are smaller than non-CF babies and why some patients with good clinical status fail to reach their growth potential. The results also suggest that measuring IGF1 levels might be of value as a biomarker to predict disease severity or the response to therapeutics. Finally, they raise the possibility that IGF1 supplementation beginning in infancy might be beneficial in CF.

These very interesting findings do explain the suboptimal growth of some CF infants despite optimal treatment and a good clinical condition. Also they provide a very plausible explanation for the well documented reduced birth weight of CF infants as a group. Various studies have shown that growth hormone has some value in improving growth of CF children (see TOPICS - ENDOCRINOLOGY) but as yet IGF-1 supplementation has not been reported although the relationship of growth and IGF-1 has been described in CF (2009 Switzer M. Rice J. Rice M. Hardin DS. Insulin-like growth factor-I levels predict weight, height and protein catabolism in children and adolescents with cystic fibrosis. J Pediatr Endocrinol 2009; 22:417-424).[PubMed]

 

 

Figure 3: Professor Jeffrey Wine

2011 Abu-El-Haija M. Sinkora M. Meyerholz DK. Welsh MJ. McCray PB Jr. Butler J. Uc A. An activated immune and inflammatory response targets the pancreas of newborn pigs with cystic fibrosis. Pancreatology 2011; 11:506-515.[PubMed]

In cystic fibrosis (CF), pancreatic disease begins in utero and progresses over time to complete destruction of the organ. Although inflammatory cells have been detected in the pancreas of humans and pigs with CF, their subtypes have not been characterized. The authors used four-color flow cytometry, and analyzed the surface antigens of leukocytes in pancreas, blood, and mesenteric lymph nodes (MLN) of newborn pigs with CF and in those without CF. Pancreatic histopathology was examined with HE stain.

The CF pig pancreas had patchy distribution of inflammatory cells with neutrophils/macrophages in dilated acini, and lymphocytes in the interstitium compared to non-CF. They discovered an activated immune response that was specific to the pancreas of newborn CF pigs; inflammation was not systemic. The presence of both innate and adaptive immune cells suggests that the disease process is complex and extensive.

 

2011 Pierucci-Alves F, Akoyev V, Stewart JC 3rd, Wang LH, Janardhan KS, Schultz BD. Swine models of cystic fibrosis reveal male reproductive tract phenotype at birth. Biol Reprod 2011; 85:442-451. [PubMed]
The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of CF pigs. Tracts from CFTR(-/-) and CFTR(dF508/dF508) neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR(-/-) monolayers can exhibit Na(+) reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Cl(-) and/or HCO(3)(-) transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTR mutations and duct abnormalities that are manifested at birth.

So the CF pigs have a similar genital tract abnormality to that present in men with CF which is attributable to the CFTR mutations (Kaplan E et al. N Eng J Med 1968; 279:65-69. Above [PubMed])

 

2011 McLachlan G, Davidson H, Holder E, Davies LA, Pringle IA, Sumner-Jones SG, Baker A, Tennant P, Gordon C, Vrettou C, Blundell R, Hyndman L, Stevenson B, Wilson A, Doherty A. Shaw DJ, Coles RL, Painter H, Cheng SH, Scheule RK, Davies JC, Innes JA, Hyde SC, Griesenbach U, Alton EW, Boyd AC, Porteous DJ, Gill DR, Collie DD. Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung. Gene Ther 2011; 18:996-1005. [PubMed]
The UK Gene Therapy Consortium report the use of a large animal model to assess three non-viral gene transfer agents (GTAs): 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Ther authors' conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as their lead GTA for clinical trials of gene therapy in CF patients.

The UK Gene Therapy Consortium's trial of gene therapy started in 2012 and the present study confirmed their choice of GL67A as the most suitable gene transfer agent to be used in the trial.. The multidose trial will represent the culmination of over 10 years work on the part of the members of the GTC in London, Oxford and Edinburgh. The presence of mild inflammation was also noticed in the first CF patients treated in the pilot study (Davies JC et al. Safety and expression of a single dose of lipid-mediated CFTR gene therapy to the upper and lower airways of patients with CF. Pediatr Pulmonol 2011; Suppl 34: Abstract 198. Page 281).

 

 

2011 Ostedgaard LS, Meyerholz DK, Chen JH, Pezzulo AA, Karp PH, Rokhlina T, Ernst SE, Hanfland RA, Reznikov LR, Ludwig PS, Rogan MP, Davis GJ, Dohrn CL, Wohlford-Lenane C, Taft PJ, Rector MV, Hornick E, Nassar BS, Samuel M, Zhang Y, Richter SS, Uc A, Shilyansky J, Prather RS, McCray PB Jr, Zabner J, Welsh MJ, Stoltz DA. The dF508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs. Science Translational Medicine 2011; 3:74ra24.[PubMed]
To better understand the effects of the dF508 mutation in vivo, we produced CFTR dF508/dF508 pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-dF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTR dF508/dF508 airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3',5'-monophosphate (cAMP) agonists were less potent at stimulating current in CFTR(dF508/dF508) epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTR dF508/dF508) pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-dF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

An important publication outlining some of the features of the recently described CF pigs. The question is often asked "How much CFTR do you need?" Well according to these findings 6% does not seem to be adequate to prevent lung disease.

 

 

2011 Chang EH, Lacruz RS, Bromage TG, Bringas P Jr, Welsh MJ, Zabner J, Paine ML. Enamel pathology resulting from loss of function in the cystic fibrosis transmembrane conductance regulator in a porcine animal model. Cells Tissues Organs 2011; 194(2-4):249-254. [PubMed]
Mice null for Cftr and mice null for Slc4a2 have enamel defects, and there are some case reports of enamel anomalies in patients with CF. In this study the authros demonstrate that both Cftr and AE2 expression increased significantly during the rat enamel maturation stage versus the earlier secretory stage (5.6- and 2.9-fold, respectively). These qPCR data im- ply that there is a greater demand for Cl(-) and bicarbonate (HCO3-) transport during the maturation stage of enamel formation, and that this is, at least in part, provided by changes in Cftr and AE2 expression. In addition, the enamel phenotypes of 2 porcine models of CF, CFTR-null, and CFTR-DF508 have been examined using backscattered electron microscopy in a scanning electron microscope. The enamel of newborn CFTR-null and CFTRl DeltaF508 animals is hypomineralized. Together, these data provide a molecular basis for interpreting enamel disease associated with disruptions to CFTR and AE2 expression.

The CF pig model providing evidence for the cause of enamel defects reported in CF patients

 


2012 Pezzulo AA, Tang XX, Hoegger MJ, Alaiwa MH, Ramachandran S, Moninger TO, Karp PH, Wohlford-Lenane CL, Haagsman HP, van Eijk M, Bánfi B, Horswill AR, Stoltz DA, McCray PB Jr, Welsh MJ, Zabner J. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature. 2012; 487(7405):109-13. [PubMed]
The authors found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO(3)(-) transport. Without CFTR, airway epithelial HCO(3)(-) secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. They suggest that these findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.


Dr Alejandro Pezzulo is on the staff of University of Iowa Carver College of Medicine and has published extensively on various aspects of cystic fibrosis both clinical and laboratory research and on CF animal models - particulalry many studies relating to CF pigs.

 

 

2013 Reznikov LR, Dong Q, Chen JH, Moninger TO, Park JM, Zhang Y, Du J, Hildebrand MS, Smith RJ, Randak CO, Stoltz DA, Welsh MJ. CFTR-deficient pigs display peripheral nervous system defects at birth. Proc Nat Acad Sci USA 2013; 110:3083-8. Free text available[PubMed]

The authors tested the hypothesis that disruption of the cystic fibrosis transmembrane conductance regulator (CFTR) gene directly influences nervous system function by studying newborn CFTR(-/-) pigs. They showed CFTR expression and activity in Schwann cells, and loss of CFTR caused ultra structural myelin sheath abnormalities similar to those in known neuropathies. The authors concluded that loss of CFTR directly alters Schwann cell function suggesting that some nervous system defects in people with cystic fibrosis are likely primary rather than secondary.

There is an extensive useful review of the neurological problems that have been reported as affecting people with CF.

 

 

2013 Stoltz DA. Rokhlina T. Ernst SE. Pezzulo AA. Ostedgaard LS. Karp PH. Samuel MS. Reznikov LR. Rector MV. Gansemer ND. Bouzek DC. Alaiwa MH. Hoegger MJ. Ludwig PS. Taft PJ. Wallen TJ. Wohlford-Lenane C. McMenimen JD. Chen JH. Bogan KL. Adam RJ. Hornick EE. Nelson GA 4th. Hoffman EA. Chang EH. Zabner J. McCray PB Jr. Prather RS. Meyerholz DK. Welsh MJ. Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs.J Clin Invest 2013; 123:2685-2693. [PubMed]

Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time.

These data indicate that expressing CFTR in intestine, without pancreatic or hepatic correction, is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

 

2013 Adam RJ. Michalski AS. Bauer C. Abou Alaiwa MH. Gross TJ. Awadalla MS. Bouzek DC. Gansemer ND. Taft PJ. Hoegger MJ. Diwakar A. Ochs M. Reinhardt JM. Hoffman EA. Beichel RR. Meyerholz DK. Stoltz DA. Air trapping and airflow obstruction in newborn cystic fibrosis piglets. Am J Resp Crit Care 2013; 188:1434-41.[PubMed]

Air trapping and airflow obstruction are being increasingly identified in infants with cystic fibrosis. These findings are commonly attributed to airway infection, inflammation, and mucus build up. This study was to determine if air trapping and airflow obstruction are present before the onset of airway infection and inflammation in cystic fibrosis. On the day they are born, piglets with CF lack airway infection and inflammation so the authors used newborn wild-type piglets and piglets with CF to assess air trapping, airway size, and lung volume with inspiratory and expiratory X-ray computed tomography scans. Micro-computed tomography scanning was used to assess more distal airway sizes. Airway resistance was determined with a mechanical ventilator. Mean linear intercept and alveolar surface area were determined using stereologic methods.

On the day they were born, piglets with cystic fibrosis exhibited air trapping more frequently than wild-type piglets (75% vs. 12.5%, respectively). Moreover, newborn piglets with CF had increased airway resistance that was accompanied by luminal size reduction in the trachea, mainstem bronchi, and proximal airways. In contrast, mean linear intercept length, alveolar surface area, and lung volume were similar between both genotypes.

The authors conclude that the presence of air trapping, airflow obstruction, and airway size reduction in newborn piglets with cystic fibrosis before the onset of airway infection, inflammation, and mucus accumulation indicates that cystic fibrosis impacts airway development. They suggest their findings suggest that early airflow obstruction and air trapping in infants with cystic fibrosis might, in part, be caused by congenital airway abnormalities.

This is a fascinating study showing what has been suspected previously – that the airways of the newborn with CF are anatomically abnormal. Anatomical abnormalities have already been described in airways of CF piglets (Meyerholz DK et al, 2010. [PubMed] ). The trachea and mainstem bronchi had a uniformly small calibre and cross-sections of trachea were less circular than in controls, the trachealis smooth muscle had an altered bundle orientation and increased transcripts in a smooth muscle gene set, the submucosal glands were hypoplastic and had global reductions in tissue-specific transcripts. To learn whether any of these changes occurred in young patients with CF, they examined CT scans from children 2 years of age and younger, and found that CF tracheas were less circular in cross-section, but lacked differences in lumen area. However, analysis of previously published morphometric data showed reduced tracheal lumen area in neonates with CF.

Occasionally the lower airway abnormalities are such that a young infant presents a clinical picture resembling tracheomalacia with intractable expiratory wheezing which gradually improves through infancy.

 

2013  Awadalla M, Miyawaki S, Abou Alaiwa MH, Adam RJ, Bouzek DC, Michalaski AS, Fuld MK, reynolds KJ, Hoffman EA, Lin CL, Stoltz DA.  "Early airway structural changes in cystic fibrosis pigs as a determinant of particle distribution and deposition" Ann Biomed Eng 2013. [PubMed]

At birth, before infection and inflammation, CF pigs have airways that are irregularly shaped and have a reduced caliber compared to non-CF pigs.  The authors hypothesized that these airway structural abnormalities affect airflow patterns and particle distribution. To test this hypothesis they used computational fluid dynamics (CFD) on airway geometries obtained by computed tomography of newborn non-CF and CF pigs. For the same flow rate, newborn CF pig airways exhibited higher air velocity and resistance compared to non-CF. Moreover they found that, at the carina bifurcation, particles greater than 5-μm preferably distributed to the right CF lung despite almost equal airflow ventilation in non-CF and CF. CFD modeling also predicted that deposition efficiency was greater in CF compared to non-CF for 5- and 10-μm particles. These differences were most significant in the airways included in the geometry supplying the right caudal, right accessory, left caudal, and left cranial lobes.

The authors suggest that the irregular particle distribution and increased deposition in newborn CF pig airways suggest that early airway structural abnormalities might contribute to CF disease pathogenesis.

 

2013 CaoH, Machuca TN, Yeung JC, Wu J, Du C, Hashimoto K, Linacre V, Coates AL, leung K, Wang J, Yeger H, Cutz E, Liu M, Keshavjee S, Hu J. Efficient gene delivery to pig airway epithelia and submucosal glands using helper-dependent adenoviral vectors.  Mol Ther Nucleic Acids 2013; 2:e127. doi: 10.1038/mtna.2013.55.[PubMed]

Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF). However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, they utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors.

Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. They found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR).

Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy

 

2013  Stoltz DA, Rokhlina T, Ernst SE, Pezzulo AA, Ostedgaard LS, Karp PH, Samuel MS, Reznikov LR, Rector MV, Gansemer ND, Bouzek DC, Alaiwa MH, Hoegger MJ, Ludwig PS, Taft PJ, Wallen TJ, Wohlford-Lenane C, McMenimen JD, Chen JH, Bogan KL, Adam RJ, Hornick EE, Nelson GA 4th, Hoffman EA, Chang EH, Zabner J, McCray PB Jr, Prather RS, Meyerholz DK, Welsh MJ. Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs.  J Clin Invest. 2013 ;123:2685-93.[PubMed]

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. The authors hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. They produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time.

The authors consider these data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. The suggest this model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

 

2014 Griffin MA, Restrepo MS, Abu-El-Haija M, Wallen T, Buchanan E, Rokhlina T, Chen YH, McCray bPBJr, Davidson BL, Divekar A, Uc A. Novel gene delivery method transduces porcine pancreatic duct epithelial cells.  Gene Ther. 2014; 21(2):123-30. [PubMed]

Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors.

The authors' objective was to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. They catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas.

Their technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages-delivery approaches that can be translated to humans.

Although modern pancreatic enzyme replacement therapy is relative effective, at present there is no way of preventing the relentless destruction of the pancreas leading to CF related diabetes. The development of approaches such as described here is welcome.

 

 

2014 Guo JJ. Stoltz DA. Zhu V. Volk KA. Segar JL. McCray PB Jr. Roghair RD. Genotype-specific alterations in vascular smooth muscle cell function in cystic fibrosis piglets. J Cyst Fibros 2014; 13(3):251-9.[PubMed] 24183914+
The authors hypothesised that compared to wild-type or CFTR-null aorta, aorta from CFTR-F508del (dF) piglets will have reduced endoplasmic reticulum calcium mobilisation and decreased vasoconstriction. Aortic reactivity was assessed by myography, and ratiometric calcium imaging was performed in isolated vascular smooth muscle cells. The aorta from dF piglets had reduced myogenic tone (P<0.001) and decreased constriction to KCl (P<0.05). Combined inhibition of ryanodine and IP3 receptors decreased wild-type and CFTR-null responses to levels seen in dF aorta. Compared to wild-type cells, dF-expressing smooth muscle cells had reduced calcium transients, while CFTR-null cells had decreased baseline intracellular calcium concentrations.
The authors concluded that expression of CFTR-F508del interferes with smooth muscle cell calcium handling and decreases aortic responsiveness.

 

2014 Hoegger MJ. Awadalla M. Namati E. Itani OA. Fischer AJ. Tucker AJ. Adam RJ. McLennan G. Hoffman EA. Stoltz DA. Welsh MJ. Assessing mucociliary transport of single particles in vivo shows variable speed and preference for the ventral trachea in newborn pigs. Proc Natl Acad Sci USA. 2014; 111(6):2355-60. [PubMed]24474805+
Mucociliary transport (MCT) is an innate defence mechanism that removes particulates, noxious material, and microorganisms from the lung. It remains uncertain whether MCT abnormalities contribute to the genesis of disease or whether they are secondary manifestations that may fuel disease progression. The authors used X-ray multidetector-row-computed tomography to track movement of individual particles in the large airways of newborn CF pigs. Multidetector-row-computed tomography imaging provided high spatial and temporal resolution and registration of particle position to airway anatomy.
The authors observed that cilia orientation directs particles to the ventral tracheal surface.and also there was substantial heterogeneity in the rate of individual particle movement; they speculate that variations in mucus properties may be responsible. The increased granularity of MCT data provided by this assay may provide an opportunity to better understand host defence mechanisms and the pathogenesis of airway disease.

 

2014 Judge EP. Hughes JM. Egan JJ. Maguire M. Molloy EL. O'Dea S. Anatomy and bronchoscopy of the porcine lung. A model for translational respiratory medicine. [Review] Am J Respir Cell Mol Biol 2014; 51(3):334-43. [PubMed]24828366+
For the respiratory medicine field, the similarities between pig and human lungs give the porcine model particular potential for advancing translational medicine. The aims of this review are to collate the disparate literature on porcine lung anatomy, histology, and microbiology; to provide a comparison with the human lung; and to describe appropriate bronchoscopy procedures for the pig lungs to aid clinical researchers working in the area of translational respiratory medicine using the porcine model.

 

 

2014 Sun X. Olivier AK. Liang B. Yi Y. Sui H. Evans TI. Zhang Y. Zhou W. Tyler SR. Fisher JT. Keiser NW. Liu X. Yan Z. Song Y. Goeken JA. Kinyon JM. Fligg D. Wang X. Xie W. Lynch TJ. Kaminsky PM. Stewart ZA. Pope RM. Frana T. Meyerholz DK. Parekh K. Engelhardt JF. Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets. J Respir Cell Mol Biol 2014; 50(3):502-12 [PubMed]24074402+
Previously, these authors described that newborn CF transmembrane conductance regulator-knockout ferrets rapidly develop lung infections within the first week of life. Here, they report a more slowly progressing lung bacterial colonisation phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of the CF ferrets.

 

2014 Awadalla M. Miyawaki S. Abou Alaiwa MH. Adam RJ. Bouzek DC. Michalski AS. Fuld MK. Reynolds KJ. Hoffman EA. Lin CL. Stoltz DA. Early airway structural changes in cystic fibrosis pigs as a determinant of particle distribution and deposition. Ann Biomed Eng 2014; 42(4):915-27.c [PubMed] 24310865+
Cystic fibrosis pigs develop lung disease similar to humans with CF. At birth, before infection and inflammation, CF pigs have airways that are irregularly shaped and have a reduced caliber compared to non-CF pigs. The authors hypothesised that these airway structural abnormalities affect airflow patterns and particle distribution. To test this hypothesis they used computational fluid dynamics (CFD) on airway geometries obtained by computed tomography of newborn non-CF and CF pigs.
The irregular particle distribution and increased deposition in newborn CF pig airways suggest that early airway structural abnormalities might contribute to CF disease pathogenesis.
 
In contrast to the cystic fibrosis mice, the pigs have airway abnormalities that are similar to those in affected infants and apparently are proving very useful in explaining certain features present in cystic fibrosis infants.

 


2015 Yan Z; Stewart ZA; Sinn PL; Olsen JC; Hu J; McCray PB Jr; Engelhardt JF.  Ferret and pig models of cystic fibrosis: prospects and promise for gene therapy.  Hum Gene Ther 2015; 26(1):38-49.   25675143[PubMed]
The disease phenotypes in the ferret and pig models is more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, the authors examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. They discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, they hope to convey the potential of these new animal models for developing CF gene and cell therapies.

 


2015 Olivier AK; Gibson-Corley KN; Meyerholz DK. Animal models of gastrointestinal and liver diseases. Animal models of cystic fibrosis: gastrointestinal, pancreatic, and hepatobiliary disease and pathophysiology. [Review] Am J Physiol - Gastr L 2015; 308(6):G459-71.  [PubMed]

Multiple organ systems, including the gastrointestinal tract, pancreas, and hepatobiliary systems, are affected by cystic fibrosis. Many of these changes begin early in life and are difficult to study in young CF patients. Recent development of novel CF animal models has expanded opportunities in the field to
better understand CF pathogenesis and evaluate traditional and innovative therapeutics. In this review, the authors discuss manifestations of CF disease in gastrointestinal, pancreatic, and hepatobiliary systems of humans and animal models. They also compare the similarities and limitations of animal models and discuss future direction for modelling CF.

 

 

2015 Uc A; Olivier AK; Griffin MA; Meyerholz DK; Yao J; Abu-El-Haija M; Buchanan KM; Vanegas Calderon OG; Abu-El-Haija M; Pezzulo AA; Reznikov LR; Hoegger MJ; Rector MV; Ostedgaard LS; Taft PJ; Gansemer ND; Ludwig PS; Hornick EE; Stoltz DA; Ode KL; Welsh MJ; Engelhardt JF; Norris AW.  Glycaemic regulation and insulin secretion are abnormal in cystic fibrosis pigs despite sparing of islet cell mass.  Clinical Science 2015; 128(2):131-42.[PubMed]
Glycaemic abnormalities and insulin secretion defects were present in newborn CF pigs and spontaneous hyperglycaemia developed over time. It is interesting that functional changes in CF pig pancreas were not associated with a decline in islet cell mass. The results suggest that functional islet abnormalities, independent of structural islet loss, contribute to the early pathogenesis of CFRD.
 


2015 Hayden UL, Carey HV. Cellular localization of cystic fibrosis transmembrane regulator protein in piglet and mouse intestine. Cell Tissue Res. 1996 Feb;283(2):209-13. [PubMed]

Antibodies raised against the cystic fibrosis transmembrane regulator protein (CFTR) were used to localize CFTR in intestinal tissues of piglets and mice. Positive staining for CFTR was detected in goblet cells of both species. A second population of epithelial cells of unknown phenotype was also labeled by anti-CFTR antibodies. The labeling pattern was abolished by preincubation of anti-CFTR antibodies with the immunogen or when non-immune IgG was used in place of anti-CFTR antibodies.
 
- These results support other studies that suggest that alterations in goblet cell function may be involved in the intestinal abnormalities associated with cystic fibrosis.